Hepatoprotective Activity of the Ethanolic Extract of Ficus caricaLinn. LeavesinCarbon Tetrachloride-Induced Hepatotoxicityin Rats.

The ethanolic extract of Ficus carica leaves was screened for hepatoprotective and antioxidant activity in hepatotoxic Albino rats induced via carbon tetrachloride. The degree of protection was measured by estimating biochemical parameters such as serum glutamate Oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase ( SGPT),totalprotein (TP), totalalbumin (TA), alkaline phosphatase (ALKP) and the level of total serum bilirubin. The extract in addition reduced CCl4 induced lipid peroxidation in-vivo and in-vitro. The ethanolic extract (50 mg/kg, 100mg/kg,200mg/kg)exhibited significant hepatoprotection incarbontetra chloride in toxicated rats in a dose dependant manner. The hepatoprotective effects of the extract were comparable with the standard drug silymarin 10)mg/kg body weight, IP).


Introduction
body, regulating homeostasis and is a frequent target for a number of toxicants (1). In spite problems are on the rise. Regrettably there are only a few drugs with serious side effects available for the treatment of liver ailments (2). In view of the undesirable side effects of synthetic agents, there is growing focus towards the therapeutic evaluation of medicinal plants using systemic research methodology.
Ficus carica commonly known as Fig is a small or moderately sized deciduous tree indigenous to asia minor, persia, syria and the mediterranean region (3). The plant leaves has been reported to contain furanocoumarins such as psoralen, bergapten, xanthotoxin (4), triterpenes such as calotropenyl acetate, lupeol acetate (5), isoschaftoside (6) and certain sterols.
Fig leaves have been traditionally used in the treatment of vitiligo, diabetes, coughs, asthma, constipation and gingivitis (3, 7). The leaves include cytotoxic (8), hypoglycemic (9) and anthelmintic activity (10). furanocoumrains and triterpens found in medicinal plants are used as hepatoprotective drugs (11), it was thought worthwhile to conduct hepatoprotective studies on the leaves of the Ficus carica to validate its use in the traditional system of medicine.

Plant material
Ficus carica were collected from Jamia Hamdard (Hamdard university) campus, New Delhi, India and authenticated by the taxonomist at the Department of Botany, Faculty of Science, Hamdard University. A voucher specimen was deposited in the herbarium of University for future reference.

Preparation of extract
Air dried and coarsely powdered leaves of the plant (1 kg) were soxhlet extracted with ethanol for 72 h. The ethanolic extract was then concentrated on a water bath and dried under reduced pressure to achieve a dark brown mass (95 g; yield-9.5%).

Preliminary phytochemical screening
On preliminary phytochemical screening using the reported method (12), the ethanolic extract of the Ficus carica leaves showed positive tests for glycosides, steroids, triterpens

Determination of phenolic compounds
The total phenols were estimated by the Folin-Ciocalteu reagent according with the method of Gao et al. (13). The total content was expressed as mg of gallic acid equivalents/g extract.
according to Kosalec et al. (14). The total content was expressed as mg of quercetin equivalents/g extract.

Animals
Hepatoprotective activity was carried out on Albino rats of either sex (110-145 g), supplied by the central animal house facility of Jamia Hamdard, New Delhi (Registration no. 173/ CPCSEA). The rats were maintained in a 12 h light/dark cycle at 25 ± 2 C. They were allowed free access to a standard pellet diet (Amrut India) and water ad libitum. The study was approved by the ethics committee CPCSEA and ethical norms were strictly followed during all experimental procedures.

Drugs and dosing schedule
The animals were divided into six groups; group I (control), group II (CCl 4 treated), group III (CCl 4 + silymarin treated), group IV, V and VI (CCl 4 + extract). Animals of groups II, III, IV, V and VI were administered 50% (v/v) CCl 4 weight per day for 4 days via the S.C. route. Simultaneously but at different hours of the day, animals of groups III, IV, V and VI were fed with silymarin suspension (10 mg/kg body weight, IP) in addition to ethanolic extract in doses of 50 mg/kg, 100 mg/kg and 200 mg/kg body weight, IP for 4 days respectively. Animals of group I kg body weight.

Serum analysis
On the day 5, after the treatment period all of the subject animals were anaesthetized and and their serum was separated by centrifugation at 3000 rpm at 30 o C for 15 min. This was subsequently analysed for various biochemical parameters including serum transaminases viz. SGOT (15), SGPT (15), total protein (16), total albumin, alkaline phosphatase (17) and total bilirubin content (18).

In-vitro antioxidant activity by DPPH method
The stable 1, 1-diphenyl-2-picryl hydrazyl determination of free radical-scavenging activity of the extract (19). 2.95 mg of DPPH extract in various concentrations was mixed in at 517 nm. The reaction mixture was set-aside in the dark at room temperature for 90 min and then its absorbance was recorded at 517 nm.
Corresponding blank readings were also taken and the remaining DPPH was calculated. The experiment was repeated three times. Ascorbic acid and quercetin were used as standard controls.

In-vivo antioxidant activity
The in-vivo antioxidant activity of the ethanolic extract was carried out in CCl 4 intoxicated rats. The liver samples collected were washed with chilled normal saline, weighed and 10% (w/v) liver homogenate was generated in ice cold 0.15 M KCl solution using was centrifuged at 2000 rpm at 4 C for 10 min and the clear supernatant was used for the estimation of the following antioxidant markers.

Estimation of liver TBARS
The measurement of Thiobarbituric acid reactive substances (TBARS) was carried out as an index of lipid peroxidation and measured in terms of malondialdehyde (MDA) content by following the method outlined by Ohkawa et al. (20). The total protein content in the tissue homogenate was determined using the method set et al. (21). The values of TBARS were presented as nmol MDA /mg protein.

Statistical analysis
The results of biochemical parameters are reported as mean ± SEM. The statistical way analysis of variance (ANOVA) followed by Dunnet's t-test (23). A p-value of < 0.05 was

Results
of Ficus carica leaves showed the presence of class of phytoconstituents as revealed by the preliminary phytochemical screening. The content was found to be 135.8 ± 7.21 mg in gallic acid equivalent/g extract and 60.5 ± 5.2 mg in quercetin equivalent/g extract, respectively.
The administration of CCl 4 hepatocellular damage as evident from the increase in serum activities of SGOT, SGPT, Alkaline addition to a decrease in the level of total protein (TP) and total albumin (TA) concentration (7.81, normal control group. Treatment of rats with the ethanolic extract of leaves at a dose of 50 mg/ kg, 100 mg/kg and 200 mg/kg body weight i.p.  (Table 2). In the liver tissues standard Silymarin at a dose of 10 mg/Kg also showed elicited by the ethanolic extract were comparable with that of the standard drug silymarin.

Discussion
CCl 4 is one of the most commonly used hepatotoxins in experimental studies of liver diseases (24). The hepatotoxic effects of CCl 4 are largely due to its active metabolite, trichloro methyl radical (25). These activated radicals bind covalently to macromolecules and induce peroxidative degradation of endoplasmic reticulum membrane lipids rich in polyunsaturated fatty acids. This leads to the formation of lipid peroxides, which in turn creates products such as malondialdehyde (MDA) that cause damage to of the bio membrane is one of the principle causes of CCl 4 toxicity, this is evidenced by the elevation of TBARS and a decrease in the activity of the free radical scavenging enzyme (GSH). In addition an elevation in the serum marker enzymes is also witnessed. The increase of jaundice. An increase in transaminases was a clear indication of cellular leakage and a loss of the functional integrity of the cell membrane the liver enzyme levels and increased the level of total serum protein in a dose dependant manner, indicating hepatoprotection.
The DPPH test is a very convenient method for screening small antioxidant molecules as the intensity of reaction can be analysed by a simple spectrophotometric method (27). The DPH radical is scavenged by antioxidants through the donation of hydrogen to form stable radical DPPH molecules. The antioxidant radicals formed are stabilized through the formation of a non-radical product.
IC 50 is considered to be a good measure of and extracts. The IC 50 of the ethanolic extract of Ficus carica be comparable to that of standard ascorbic acid and quercetin.
biomembrane is one of the principle causes of CCl 4 toxicity. This is evidenced by the elevation of TBARS and a decrease in the activity of free radical scavenging enzymes such as GSH in CCl 4 intoxicated animals. Glutathione is Values are mean ± SEM (n = 6), * p < 0.001 Vs Control ** p < 0.05; *** p < 0.001 Vs CCl 4 . One-way analysis followed by Dunnet's t-test. TBARS: Thiobarbutric acid reactive substances, GSH: Reduced glutathione. a naturally occurring tripeptide and a nonenzymatic biological antioxidant that is abundant in many living creatures. It is widely known that can lead to tissue injury and disorder. The increase in the level of MDA in liver induced by CCl 4 suggests enhanced lipid peroxidation. This leads to tissue damage and the failure of antioxidant defence mechanisms preventing the formation of excessive free radicals. Treatment with the ethanolic extract (50, 100 and 200 mg/Kg) reduced the elevated level of TBARS (Thobarbituric acid reactive substances) in CCl 4 intoxicated liver tissues. It was also observed that GSH depletion due to the CCl 4 challenge was reversed by the test extract.
Preliminary phytochemical screening glycosides. The free radical mediated process has been implicated in the pathogenesis of different diseases. Thus the antioxidant activity of the ethanolic extract may be due to the Its hepatoprotective activity is attributed to its antioxidant properties. Further isolation of the active principles responsible for its hepatoprotective activity is currently in progress within our laboratory. work.